Subject: Isolating High MW DNA from Gel- Best Way??
From: Peter T. Boag, BOAGP at QUCDN.QUEENSU.CA
Date: Thu, 10 Mar 1994 17:31:03 GMT
In article <CMGLzs.Isz at knot.ccs.queensu.ca> Peter T. Boag,
BOAGP at QUCDN.QUEENSU.CA writes:
>We are doing some genomic library building and want to
>preferentially clone fragments in the 5 to 15 kb range. We have
>experimented with various ways to isolate this range of DNA from an
>Agarose gel, and always seem to end up with a low yield. We have
>tried inserting a piece of dialysis membrane and running the DNA
>onto it. We have cut a well in the gel ahead of the desired area,
>then pipet off the DNA as it runs into the well. We are thinking
>now of excising the relevant piece of gel and spinning it above a
>siliconized glass wool plugged eppindorf with a hole in the bottom
>inserted into a second eppindorf, and collecting the runoff (this is
>a variant of the "freeze and squeeze" approach).
>>Any suggestions for this type of isolation, either quit and dirty or
>fancy commercial approaches such as these electroelution chambres??
I always found GeneClean from BIO 101 to be the ultimate in DNA
recovery from agarose, I have used it or a variant for many years.
Recently I have had problems with the latest version, GeneClean II,
but this seems to be due to the High Salt buffer going off after 6
to 12 months, buying a new kit when this occurs clears up the
problem. The principle is that saturated NaI (or perchlorate in
some systems) both melts the agarose at elevated temperature and
(due to high salt) causes the DNA to adhere to powdered glass (or
similar in other kits). the DNA/Glass is washed extensively and
then the DNA eluted off with low salt buffer or water.
The kit can be used for recovery of DNA from agarose, de-salting,
removing eg restriction enzymes prior to ligation, cleaning up PCR
products and curing the common cold, try it and see.
Tony
.
Tony P Hodge
Structural Studies Division
Medical Research Council Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 2QH
UK
Phone (0223) 402260
Fax (0223) 213556